The purpose of this investigation is to study the effects of the protein glutamine synthetase on the visible spectrum of the dye Cibacron Blue F3GA. A dye difference spectrum with the native (taut) form of glutamine synthetase is characterized by a maximum at 640 nm and a minimum at 700 nm. The spectra obtained when the dye binds to the relaxed and subunit forms of glutamine synthetase (two known structural forms of the protein) differ from each other and clearly differ from the characteristic spectrum obtained with the native (taut) GS. Purification of the dye resulted in the separation of three chromatographically distinct subfractions of Cibacron Blue. The difference spectra obtained when each of these subfractions binds to taut, relaxed forms of GS clearly differ from each other. These results indicate that dye difference spectroscopy can be used to monitor changes in protein conformation associated with the relaxation and subunit dissociation reactions.